Role of host cell polarity and leading edge properties in Pseudomonas type III secretion

Abstract

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration. INTRODUCTIONOriginally identified because of its role in Yersinia virulence (Cornelis et al., 1989), type III secretion (T3S) is now recognized to contribute to the pathogenesis of a large number of Gram-negative bacteria. T3S allows the direct translocation of 'effectors' from the bacterial cytosol into eukaryotic cells, enabling bacteria to manipulate host cells to establish infections while evading immune responses. The T3S system includes a bacterially formed 'injectisome' needle-like nanostructure that serves as a conduit for transferring bacterial effectors to eukaryotic cells. A bacterially formed 'translocon' channel is then believed to mediate effector translocation across host cell membranes. The mechanism underlying T3S translocon channel formation and host involvement in this process remain the least understood events in T3S. We have used the Abbreviations: CTB, cholera toxin B subunit; dsPFO, prepore locked PFO; HA, haemagglutinin; IF, immunofluorescent/immunofluorescence; IQGAP1, IQ-motif-containing GTPase-activating protein 1; LatB, latrunculin B; LE, leading edge; MbCD, methyl-beta-cyclodextrin; MT, microtubule; Pa-ExoS-HA, P. aeruginosa stra...

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…Thus, infection studies with P. aeruginosa revealed that some cell lines are resistant against T3S-mediated protein injection, indicating the requirement of certain host cell properties for efficient effector protein translocation (357,471). This observation was supported by the finding that the alteration of the host plasma membrane composition renders cells insensitive against T3S by P. aeruginosa (55). It was already previously proposed that the formation of a functional T3S translocon occurs preferentially in specific microdomains of the host cell membrane that are rich in cholesterol and glycosphingolipids (490).…”

Examining the Role of Actin-Plasma Membrane Association in Pseudomonas aeruginosa Infection and Type III Secretion Translocation in Migratory T24 Epithelial Cells

“…Consistent with actin playing a role in T3S translocation, studies of Yersinia, Shigella, and enterohemorrhagic Escherichia coli have implicated a role of actin in enhanced T3S effector translocation (41,42,43,64), but the underlying mechanism remains unknown. Evidence that the leading edge and actin might be involved in P. aeruginosa T3S translocation was initially obtained in studies of HT-29 epithelial cells, where Rac1, Cdc42, and actin polymerization at the leading edge were found to be required for P. aeruginosa T3S translocation (5). A potential role of host actin-plasma membrane association at the leading edge in T3S translocation was recognized in studies of both HT-29 and HL-60 cells, where cellular adherence to matrix (which stabilizes actin-plasma membrane associations) was found to correlate with T3S translocation efficiency (5).…”

“…T24 cells were seeded in chamber slides (Nalge Nunc International, Rochester, NY) 24 h prior to coculture for 1.5 h with P. aeruginosa expressing ExoS-WT or the pUCP plasmid control. Following coculture, T24 cells were washed twice in Dulbecco's phosphate-buffered saline (DPBS; HyClone), fixed in 3% paraformaldehyde in actin stabilization buffer (ASB) (5,26), permeabilized with 0.2% Triton X-100 in ASB, and blocked with 0.8% BSA, 0.1% fish gelatin, and 10 g/ml goat IgG. Cells were stained for F-actin by tetramethyl rhodamine isocyanate (TRITC)-phalloidin (Sigma-Aldrich), for P. aeruginosa by anti-P. aeruginosa lipopolysaccharide (LPS) (obtained from Joseph Lam, University of Guelph, Guelph, Ontario), and for trailing edge by anticaveolin-1 (N-20; Santa Cruz Biotechnology, Santa Cruz, CA).…”

“…Translocated effectors then allow bacteria to manipulate host cell processes in a manner specific to the lifestyle of the bacteria. The integral role of T3S in the establishment of P. aeruginosa infection is supported by the findings that cellular susceptibility to P. aeruginosa infection parallels cellular sensitivity to T3S and that immunity induced against the T3S translocon protein, PcrV, protects against P. aeruginosa infection (5,41,52,55).…”

“…Both cell culture models also have the potential to become sensitive to P. aeruginosa infections and T3S: in epithelial monolayers, sensitivity occurs in conjunction with disruption of apical-basolateral polarity, and in HL-60 cells, sensitivity is induced by differentiation with phorbol esters (41,52). Previous studies in our laboratory characterizing host cell properties that influence the establishment of P. aeruginosa infections identified a relationship between the leading edge of migrating eukaryotic cells and cellular sensitivity to T3S-translocated ExoS (5). Cell membranes are structured to resist pore formation, as occurs during T3S translocation.…”